| Background: Osteoarthritis（OA）,as an incurable heterogeneous disease,brings great inconvenience to the work and life of patients.Symptoms cannot be cured,and some treatments may cause secondary damage.Intra-articular injection of homologous or heterologous platelet-rich plasma（PRP）is a new treatment method for osteoarthritis,and its effectiveness has been confirmed in clinical practice,but the specific mechanism of PRP repairing articular cartilage remains unclear.Since a series of operations,such as the preparation and preservation of PRP have not formed standardized regulations,the PRP products obtained through commercial channels are quite different.The influencing factors of PRP composition include: individual factors,temperature,p H,preparation method,anticoagulant,blood sampling factors,etc.From the source of PRP preparation,exercise is a non-medication way to change blood components,stress and adapt to the body,and migration to PRP will also have some important effects on its composition and efficacy.Objective: Firstly,an acute exercise model was established to investiagte the effects of acute exercise and different recovery time on the metabolites in PRP.Secondly,a mouse model of knees osteoarthritis was established to explore the efficacy of PRP obtained after acute exercise on knees osteoarthritis in mice,and to explore the mechanism by which PRP repaired knees cartilage.Methods: 48 four-week-old male C57BL/6J mice and 20 SD rats were purchased.After rearing for 2 weeks,the mice were randomly divided into groups after adaptation,and two parts of experiments were carried out.In the first part of experiments,randomly,24 mice were divided into 4 groups and each group contains 6 mice.（1）Group S: sacrificed at rest;（2）Group R0: sacrificed immediately after exercise;（3）Group R60: sacrificed 1 hour after exercise;（4）Group R24: sacrificed 24 hours after exercise.The exercise method adopts incremental load treadmill exercise.In the first part of experiments,after blood was drawn from the eyeballs of the mice,PRP was prepared from the serum of the mice in the S,R0,R60,and R24 groups for non-target metabolomics testing to explore the changes of PRP metabolites after different rest periods after exercise.In the second part of experiments,20 rats were randomly divided into 2 groups as PRP donors,each group contains 10 rats:（1）Group DR: exercise group;（2）Group DS: sedentary group.The exercise method still adopts the incremental load treadmill exercise.Blood was collected from the eyeball immediately after exercise to prepare PRP for the treatment of osteoarthritis in OA mice.24 mice were randomly divided into 4 groups as OA treatment recipients,each group contains 6 mice:（1）Group CON: control group;（2）Group KON: treatment with iodoacetic acid;（3）Group KS: treatment with iodioacetic acid + DS-PRP;（4）Group KR: treatment with iodoacetic acid + DR-PRP.Modeling and treatment were performed simultaneously on the left and right knee joints of the hind limbs of mice.After 4 weeks of PRP treatment,serum related inflammatory factors were detected by ELISA in KON,CON,KS,and KR groups,some knee cartilages were stained to observe cartilage morphology,and PCR and WES were used to detect some biomarkers for cartilage biogenesis.Result:（1）GC-MS metabolomics analysis showed that the metabolites in PRP changed continuously at different recovery times after exercise.Compared with the resting control group,the difference was significant immediately after exercise and 24 hours after recovery（P<0.05）.Among them,the R0 group was significantly different.Compared with the S group,7 differential metabolites were up-regulated and 13 were down-regulated,and the differential metabolic pathways involved aminoacyl-t RNA biosynthesis,linoleic acid metabolism,cyanoamino acid metabolism,etc.;compared with the S group,the R24 group up-regulated 4 differential metabolites and down-regulated 40 differential metabolites.The differential metabolic pathways involved aminoacyl-t RNA biosynthesis, cyanoamino acid metabolism,glycine,serine and threonine metabolism,arginine and proline metabolism,etc.（2）Morphological results showed that the morphology of chondrocytes in the KON mice in the modeling group was significantly damaged compared with that in the control group.The cartilage in KS and KR were significantly repaired after PRP treatment.The differences in cell morphology were small.（3）Serum TGF-β was significantly increased after PRP treatment（P<0.05）,and the serum inflammatory cytokines NLRP3 and TNF-α were also significantly increased after treatment（P<0.01）.There was no significant difference between KS and KR groups（P>0.05）.（4）There was no significant difference in chondrogenesis-related gene expression among all groups,and the expression difference within the group was high.（5）WES analysis showed that there was no significant difference in protein levels of PTEN,p-Akt/Akt and PTP1 B among all groups,and the difference within the group was relatively high.Conclusion:（1）The composition of PRP metabolites changed due to different exercise recovery times.The PRP metabolome changed significantly immediately after or 24 hours of recovery after acute exercise,but there was no significant change in metabolome levels after 60 minutes of recovery.Hence,the timing of blood collection when improving PRP preparation by exercise is worth considering.（2）Compared with PRP prepared by collecting blood at rest,PRP prepared by blood collected immediately after exercise has no significant advantage in OA treatment.Whether PRP collection at other recovery time points after exercise should be further examined.（3）Rat exogenous PRP has a significant therapeutic effect on mouse osteoarthritis,but it does not show inflammation reduction,as well as gene and protein expression changes related to chondrogenesis,indicating that the repair of articular cartilage during exogenous PRP treatment is not related to inflammation level and PTEN/Akt signaling pathway. |
